In recent years, as a result of concerns related to energy supplies for transportation, as well as other environmental problems such as global warming and aerial pollution, the development of alternative energy sources to oil has become an extremely important issue. Plant biomass is the most plentiful renewable energy source on earth, and holds great promise as an alternative energy source to oil. The main component of plant biomass is lignocellulose, which is composed of polysaccharides such as celluloses and hemicelluloses (including xylan, arabinan and mannan), and lignin. These polysaccharides are hydrolyzed by a large variety of glycoside hydrolases to form monosaccharides such as glucose and xylose, which can then be used as biofuels or the raw materials for chemical products.
Lignocellulose is recalcitrant due to its highly complex structure, and is difficult to degrade or hydrolyze with a single enzyme. Accordingly, among the various polysaccharides, hydrolysis of cellulose generally requires three types of glycoside hydrolase enzymes, namely an endoglucanase (endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21). On the other hand, the structure of hemicellulose varies depending on the plant, and for example in the case of hardwoods and herbaceous plants, xylan is the main structural component. Hydrolysis of xylan requires a xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and a β-xylosidase (3.2.1.37). β-xylosidase is a hydrolase involved in the process of hydrolyzing the oligosaccharides, which are generated by hydrolysis of the hemicellulose by xylanase, to produce monosaccharides.
In conventional bioethanol production using lignocellulose as a starting resource, hydrolysis processes using high solid loading (30 to 60% solid loading) have been tested with the aim of achieving a more energy-efficient conversion to ethanol. However, in this type of lignocellulose enzymatic hydrolysis using high solid loading, the viscosity of the hydrolyzed biomass solution is high, and the hydrolysis reaction of the lignocellulose tends to proceed poorly. Accordingly, by using a thermostable enzyme and performing the enzymatic hydrolysis process at a high temperature, for example 80° C. or higher, the rate of the hydrolysis reaction can be increased, and the viscosity of the hydrolyzed biomass solution can be reduced, which is expected to enable a shortening of the hydrolysis reaction time and a reduction in the amount of enzyme required. As a result, for all of the various glycoside hydrolases, the development of enzymes having superior thermal stability is very desirable.
Many thermostable glycoside hydrolases have been obtained by isolating and identifying thermophilic microorganisms that exist in high-temperature environments, cloning genes from these isolated and cultured microorganisms, determining the DNA sequence, and then expressing the DNA using E. coli or filamentous fungi or the like. For example, Patent Document 1 discloses a β-xylosidase derived from a filamentous fungus. Patent Document 2 discloses a β-xylosidase derived from the filamentous fungus Aspergillus oryzae, the β-xylosidase exhibiting enzymatic activity at 30° C. Patent Document 3 discloses a β-xylosidase derived from Alicyclobacillus acidocaldarius, the β-xylosidase exhibiting enzymatic activity at pH 5.5 or lower and temperatures of 50° C. or higher. Patent Document 4 discloses a β-xylosidase derived from Acremonium cellulolyticus, the β-xylosidase exhibiting enzymatic activity at 45° C. Further, Non-Patent Documents 1 to 6 disclose β-xylosidases isolated from specific bacteria and filamentous fungi, including β-xylosidases having an optimum temperature in the vicinity of 60° C. In addition, Non-Patent Document 7 reports a thermostable 1-xylosidase having an optimum temperature of 95° C. However, although the catalytic efficiency Kcat/Km of the enzyme when p-nitrophenyl-β-D-xylopyranoside (hereafter often abbreviated as PNPX) is used as a substrate is an extremely high value of 1173.4 mM−1s−1, the half life of the PNPX degradation activity at 90° C. is about 30 minutes, indicating that the thermal stability is insufficient.